Immunodetection of Dichlorodiphenyltrichloroethane and its biodegradation by microbial consortium

Abstract

1, 1, 1- trichloro- 2, 2- bis- (4- chlorophenyl) ethane (DDT) is one of newlinethe highly persistent and recalcitrant organochlorine pesticides synthesized newlineand used by man. This pesticide has been listed as one of the highly toxic newlinechemical by EPA. It is shown to have mutagenic, carcinogenic and teratogenic effects on man. With continuous usage, the pesticide enters the human body via food chain. Residues of DDT have been detected in soil, water and air. Residues of DDT are generally analyzed by thin layer chromatography, gas chromatography which are expensive, labour intensive, require extensive clean up and are nor suited for on field assay. Immunoassay is highly sensitive, specific, on- field method, does not need sample clean up and has high throughput of samples. In our lab we attempted to develop an immunoassay for the detection of DDT by using both rabbit and avian antibodies. DDT levels up to 1 ng could be detected by Dot-ELISA. By signal amplification technique the detection limit could by newlineimproved to 1 pg level. A microbial consortium capable of degrading DDT was isolated in the laboratory by long term enrichment. The consortium was made of 10 bacterial isolates of which 7 belonged to Pseudomonas sp. and one each of Flavobacterium , Vibrio and Burkholderia sp. Conditions were optimized by response surface methodology. The maximum predicted percentage degradation of 96.69, 96.37, 97.97, 92.44 and 98.19 was obtained respectively for 5, 10, 20, 30 and 35 ppm initial DDT concentrations at different pH levels 7.82, 6.59, 6.92, 7.06 and 8.00 with an incubation temperature of 25 0C and inoculum concentration of 1500 mg protein/ mL. The degradation of DDT by the microbial consortium increased with time. The degradation reached 95% by 72 h of incubation. Primers designed for identifying DDT-dehydrohalogenase genes gave positive amplification with genomic DNA and plasmid DNA of Flavobacterium sp. The PCR product of newlinegenomic DNA of Flavobacterium sp. was ligated to pTZ57R/T vector and cloned in to E.coli DH5a.